Moraxella (Branhamella) catarrhalis antigens

ABSTRACT

The present invention relates to polypeptides of  Moraxella ( Branhamella )  catarrhalis  which may be useful for prophylaxis, diagnosis and/or therapy purposes. 
     
       
         
               
               
               
             
                 1 
                 
                   ATGGACACTG ACATGAAACA TTTAACAAAA CATCGCCTAT 
                 
                   
               
                   
               
                   
                 
                   CAGCTGCCAT 
                 
               
                   
               
                 51 
                 
                   CATTGGCGTT TTATTATTCA TTAGCCCATC AGTGCAAGCA 
                 
               
                   
               
                   
                 
                   AATACGACAC 
                 
               
                   
               
                 101 
                 ACCATCACAC GCTAACCAGT AGCGAGCTTA AACTTGCTGA 
               
                   
               
                   
                 TGATAGTATT 
               
                   
               
                 151 
                 ATTGATAGTA TCAATCAATT GGGTGAGCTG ACCGTCAATA 
               
                   
               
                   
                 TTCCAAATAC 
               
                   
               
                 201 
                 ACAATATTTT CAAACCAACA ACGGTGTGAG CGTTGCTTTT 
               
                   
               
                   
                 ACGCCATTAC 
               
                   
               
                 251 
                 ATGAGCTGCC TATTGTCGAT ATCAGCTTGT ATTTTAATGC 
               
                   
               
                   
                 AGGGTCAGCG 
               
                   
               
                 301 
                 TATCACCATC AGGTTGGCAA ATCAGGCACG GCTAACATGG 
               
                   
               
                   
                 TTGCAACCAT 
               
                   
               
                 351 
                 GCTCACCCAA GGAACTGACA GCCTTTCTGA AGATGAGTTT 
               
                   
               
                   
                 GTTGCTGCCA 
               
                   
               
                 401 
                 AAGAGCGTCT TCGCATTCAT TTTACCAGTA CAGCAAATAA 
               
                   
               
                   
                 GGATAACTTA 
               
                   
               
                 451 
                 ACTTTATCAT TAAGAAGCTT GTCTGATCAA TCATTATTAA 
               
                   
               
                   
                 ATCAAGCCGC 
               
                   
               
                 501 
                 CGATTTAATG GTCGATGCTG TCACTCAACC TGCTTTTGAT 
               
                   
               
                   
                 GATAAGACTC 
               
                   
               
                 551 
                 TACAACGCAA CAAAAATCAG CTCATCACCA GTTTAAAACA 
               
                   
               
                   
                 AAAAAAGCAA 
               
                   
               
                 601 
                 AACCCTTATC ATGTAGCTTC TGTTGCTTAT CATCAAGCCG 
               
                   
               
                   
                 TATATGAAAA 
               
                   
               
                 651 
                 TCATCCTTAT GCACACGCAA CCACAGGCGA TGAAGATAGT 
               
                   
               
                   
                 ATTGCCAAAA 
               
                   
               
                 701 
                 TTGATCGTGA TGAGCTGCTT AATTTTTGGC ATACTTTTAT 
               
                   
               
                   
                 TAATGCAAAT 
               
                   
               
                 751 
                 AATGCGACAC TGCTGATTAC AGGTGATATG ACCGCCGAGC 
               
                   
               
                   
                 AAGCCAAATC 
               
                   
               
                 801 
                 ACTTGCCAAC CATCTGACCG CCAAATTACC CACAGGCAAG 
               
                   
               
                   
                 TCGTATAAAA 
               
                   
               
                 851 
                 ATACGCTGGA TTTGACAAAA CCAGTTAAGG CTCGTCATAT 
               
                   
               
                   
                 CCATATTCCT 
               
                   
               
                 901 
                 CACAACAGTA GTCAAACCCA AATCATCATC GGTCATCCCA 
               
                   
               
                   
                 CCAGTAAAGT 
               
                   
               
                 951 
                 ACGCACGGAC AAAGCAGGTC GTCAAGAGTT CAGCGATTTT 
               
                   
               
                   
                 TCATTAGCTA 
               
                   
               
                 1001 
                 ATGAAATTTT GGCAGGTGGT GATTTTAATG CCAGATTGAT 
               
                   
               
                   
                 GAAAACCATT 
               
                   
               
                 1051 
                 CGAGAGCAAA AAGGCTACAC TTATGGCATT TATGGCGGTA 
               
                   
               
                   
                 TGGAACGCCT 
               
                   
               
                 1101 
                 CAGAGCAGGT GGTAATTATG TGGTTGAATT TTCAACCGAT 
               
                   
               
                   
                 GGCGATAAAG 
               
                   
               
                 1151 
                 CAGCCGATGC CATTTTAGAG ACGCTACACA TCATTAATCA 
               
                   
               
                   
                 GTCGCTGAAT 
               
                   
               
                 1201 
                 GAAGGCATAA CCCAAGAAGA GCTTGAGTTG GTGCGTTTGG 
               
                   
               
                   
                 GCAATAAAAA 
               
                   
               
                 1251 
                 TGGTTTTGCC AATATTTTTT CAAGCAATGC CAGTATTCAT 
               
                   
               
                   
                 CGTGTCATTC 
               
                   
               
                 1301 
                 GTGCTTTATT TGTTGCCGAT TATCCAAAAG ATCATCTTAA 
               
                   
               
                   
                 CCATACGCTC 
               
                   
               
                 1351 
                 AATCGCTTGG ATAATGCCAC GATAAATAGT GTTAATACCG 
               
                   
               
                   
                 CACTGAACTT 
               
                   
               
                 1401 
                 GCGTATCAAG CCTGATGAAT TTATCATCAT CACCGTGGGT 
               
                   
               
                   
                 AAAACTAAGC 
               
                   
               
                 1451 
                 CAAATTTGGA CAAATAA

RELATED PATENT APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/290,653, filed May 15, 2001, which is hereby incorporated byreference.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 484112_(—)425USPC_SEQUENCE_LISTING.txt. The textfile is 46 KB, was created on Oct. 10, 2007, and is being submittedelectronically via EFS-Web.

FIELD OF THE INVENTION

The present invention is related to polypeptides, more particularlypolypeptides of Moraxella (Branhamella) catarrhalis which may be used toprevent, diagnose and/or treat Moraxella (Branhamella) catarrhalisinfection.

BACKGROUND OF THE INVENTION

Moraxella (Branhamella) catarrhalis is a Gram-negative diplococcus thatcauses respiratory tract infections in humans. M. catarrhalis is nowaccepted as the third most common cause of otitis media in infants andchildren, after Streptococcus pneumoniae and Haemophilus influenzae. M.catarrhalis has also been associated with several other types ofinfection, including sinusitis, persistent cough, acute laryngitis inadults, suppurative keratitis, conjunctivitis neonatorum, and invasivediseases in the immunocompromised host.

Since approximately 90% of M. catarrhalis strains are resistant toantibiotics (β-lactamase positive) and that recurrent otitis media isassociated with high morbidity, there is a need for the development of avaccine that will protect hosts from M. catarrhalis infection. Aninfection by M. catarrhalis induces an immune response against antigensfound at the surface of the bacterial cells. However, many of thesesurface proteins are still not characterized, nor has the immuneresponse resulting in protection from infection by different strainsbeen determined.

To develop a vaccine that will protect hosts from M. catarrhalisinfection, efforts have mainly been concentrated on outer membraneproteins such as the high-molecular-mass protein named ubiquitoussurface protein A (UspA). This protein is considered a promising vaccinecandidate because a monoclonal antibody and polyclonal antibodies wereboth shown to be bactericidal and protective in the murinepulmonary-clearance model. However, this protein was shown to be highlyvariable among the different strains of M. catarrhalis. In addition tothis protein, other M. catarrhalis proteins have generated interest aspotential vaccine candidates. The transferrin-binding protein whichpossesses conserved epitopes exposed on the bacterial surface. However,there was divergence in the degree of antibody cross-reactivity with theprotein from one strain to another. Other investigators have alsofocused on the 45-kDa protein CD (OMP CD). This protein is highlyconserved among strains of M. catarrhalis, however adults with chronicobstructive pulmonary disease show variability in the immune responseagainst the OMP CD.

Therefore there remains an unmet need for M. catarrhalis polypeptideswhich may be used to prevent, diagnose and/or treat Moraxella(Branhamella) catarrhalis infection.

SUMMARY OF THE INVENTION

According to one aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide having at least 70% identity to asecond polypeptide comprising a sequence chosen from SEQ ID Nos: 2, 4,6, 8, 10, 12, 14 or fragments or analogs thereof.

According to one aspect, the present invention relates to polypeptidescomprising a sequence chosen from SEQ ID No: 2, 4, 6, 8, 10, 12, 14 orfragments or analogs thereof.

In other aspects, there are provided polypeptides encoded bypolynucleotides of the invention, pharmaceutical compositions, vectorscomprising polynucleotides of the invention operably linked to anexpression control region, as well as host cells transfected with saidvectors and processes for producing polypeptides comprising culturingsaid host cells under conditions suitable for expression.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the DNA sequence of BVH-MC2 gene from M. catarrhalisstrain ETSU C-2; SEQ ID NO: 1. The underlined portion of the sequencerepresents the region coding for the leader peptide.

FIG. 2 represents the amino acid sequence of BVH-MC2 polypeptide from M.catarrhalis strain ETSU C-2; SEQ ID NO: 2. The underline sequencerepresents the 30 amino acid residues leader peptide.

FIG. 3 represents the partial DNA sequence of BVH-MC2 gene from M.catarrhalis strain ETSU 658; SEQ ID NO: 3.

FIG. 4 represents the partial amino acid sequence of BVH-MC2 polypeptidefrom M. catarrhalis strain ETSU 658; SEQ ID NO: 4.

FIG. 5 represents the partial DNA sequence of BVH-MC2 gene from M.catarrhalis strain ETSU T-25; SEQ ID NO: 5.

FIG. 6 represents the partial amino acid sequence of BVH-MC2 polypeptidefrom M. catarrhalis strain ETSU T-25; SEQ ID NO: 6.

FIG. 7 represents the partial DNA sequence of BVH-MC2 gene from M.catarrhalis strain M-12; SEQ ID NO: 7.

FIG. 8 represents the partial amino acid sequence of BVH-MC2 polypeptidefrom M. catarrhalis strain M-12; SEQ ID NO: 8.

FIG. 9 represents the DNA sequence of BVH-MC3 gene from M. catarrhalisstrain ETSU C-2; SEQ ID NO: 9. The underlined portion of the sequencerepresents the region coding for the leader peptide.

FIG. 10 represents the amino acid sequence of BVH-MC3 polypeptide fromM. catarrhalis strain ETSU C-2; SEQ ID NO: 10. The underline sequencerepresents the 46 amino acid residues leader peptide.

FIG. 11 represents the DNA sequence of BVH-MC4 gene from M. catarrhalisstrain ETSU C-2; SEQ ID NO: 11. The underlined portion of the sequencerepresents the region coding for the leader peptide.

FIG. 12 represents the amino acid sequence of BVH-MC4 polypeptide M.catarrhalis strain ETSU C-2; SEQ ID NO: 12. The underline sequencerepresents the 42 amino acid residues leader peptide.

FIG. 13 represents the DNA sequence of BVH-MC5 gene from M. catarrhalisstrain ETSU C-2; SEQ ID NO: 13. The underlined portion of the sequencerepresents the region coding for the leader peptide.

FIG. 14 represents the amino acid sequence of BVH-MC5 polypeptide M.catarrhalis strain ETSU C-2; SEQ ID NO: 14. The underline sequencerepresents the 60 amino acid residues leader peptide.

FIG. 15 depicts the comparison of the partial nucleotide sequences (SEQID NO:35, 3, 5 and 7) of the BVH-MC2 genes from ETSU C-2, ETSU 658, ETSUT-25, and M-12 M. catarrhalis strains, respectively, by using theprogram Clustal W from MacVector sequence analysis software (version6.5). Underneath the alignment, there is a consensus line where * andblank spaces respectively represent identical nucleotides anddifferences between sequences.

FIG. 16 depicts the comparison of the predicted amino acid sequences(SEQ ID NO:36, 4, 6, and 8) of the BVH-MC2 partial open reading framesfrom ETSU C-2, ETSU 658, ETSU T-25, and M-12 M. catarrhalis strains,respectively, by using the program Clustal W from MacVector sequenceanalysis software (version 6.5). Underneath the alignment, there is aconsensus line where * characters represent identical amino acidresidues.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides purified and isolated polynucleotides,which encode Moraxella polypeptides which may be used to prevent,diagnose and/or treat Moraxella infection.

According to one aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide having at least 70% identity to asecond polypeptide comprising a sequence chosen from SEQ ID NO: 2, 4, 6,8, 10, 12, 14 or fragments or analogs thereof.

According to one aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide having at least 80% identity to asecond polypeptide comprising a sequence chosen from SEQ ID NO: 2, 4, 6,8, 10, 12, 14 or fragments or analogs thereof.

According to one aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide having at least 95% identity to asecond polypeptide comprising a sequence chosen from SEQ ID NO: 2, 4, 6,8, 10, 12, 14 or fragments or analogs thereof.

According to one aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide having at least 70% identity to asecond polypeptide comprising a sequence chosen from SEQ ID NO: 2, 4, 6,8, 10, 12 or 14.

According to one aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide having at least 80% identity to asecond polypeptide comprising a sequence chosen from SEQ ID NO: 2, 4, 6,8, 10, 12 or 14.

According to one aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide having at least 95% identity to asecond polypeptide comprising a sequence chosen from SEQ ID NO: 2, 4, 6,8, 10, 12 or 14.

According to one aspect, the present invention relates to polypeptidescomprising a sequence chosen from SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14 orfragments or analogs thereof.

According to one aspect, the present invention relates to polypeptidescharacterized by the amino acid sequence comprising SEQ ID NO: 2, 4, 6,8, 10, 12 or 14.

According to one aspect, the present invention provides a polynucleotideencoding an epitope bearing portion of a polypeptide comprising asequence chosen from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or fragments oranalogs thereof.

According to one aspect, the present invention provides a polynucleotideencoding an epitope bearing portion of a polypeptide comprising asequence chosen from SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14.

According to one aspect, the present invention relates to epitopebearing portions of a polypeptide comprising a sequence chosen from SEQID NO: 2, 4, 6, 8, 10, 12, 14 or fragments or analogs thereof.

According to one aspect, the present invention relates to epitopebearing portions of a polypeptide comprising a sequence chosen from SEQID NO: 2, 4, 6, 8, 10, 12 or 14.

According to one aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide having at least 70% identity to asecond polypeptide comprising a sequence chosen from SEQ ID NO: 2, 4, 6,8, 10, 12 or 14.

According to one aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide having at least 80% identity to asecond polypeptide comprising a sequence chosen from SEQ ID NO: 2, 4, 6,8, 10, 12 or 14.

According to one aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide having at least 90% identity to asecond polypeptide comprising a sequence chosen from SEQ ID NO: 2, 4, 6,8, 10, 12 or 14.

According to one aspect, the present invention provides an isolatedpolynucleotide encoding a polypeptide having at least 95% identity to asecond polypeptide comprising a sequence chosen from SEQ ID NO: 2, 4, 6,8, 10, 12 or 14.

According to one aspect, the present invention relates to polypeptidescomprising a sequence chosen from SEQ ID No: 2, 4, 6, 8, 10, 12 or 14.

According to one aspect, the present invention provides an isolatedpolynucleotide comprising a polynucleotide chosen from:

(a) a polynucleotide encoding a polypeptide having at least 70% identityto a second polypeptide comprising a sequence chosen from: SEQ ID NO: 2,4, 6, 8, 10, 12, 14 or fragments or analogs thereof;

(b) a polynucleotide encoding a polypeptide having at least 80% identityto a second polypeptide comprising a sequence chosen from: SEQ ID NO: 2,4, 6, 8, 10, 12, 14 or fragments or analogs thereof;

(c) a polynucleotide encoding a polypeptide having at least 95% identityto a second polypeptide comprising a sequence chosen from: SEQ ID NO: 2,4, 6, 8, 10, 12, 14 or fragments or analogs thereof;

(d) a polynucleotide encoding a polypeptide comprising a sequence chosenfrom: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or fragments or analogs thereof;

(e) a polynucleotide encoding a polypeptide capable of generatingantibodies having binding specificity for a polypeptide comprising asequence chosen from: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or fragments oranalogs thereof;

(f) a polynucleotide encoding an epitope bearing portion of apolypeptide comprising a sequence chosen from SEQ ID NO: 2, 4, 6, 8, 10,12, 14 or fragments or analogs thereof;

(g) a polynucleotide comprising a sequence chosen from SEQ ID NO: 1, 3,5, 7, 9, 11, 13 or fragments or analogs thereof;

(h) a polynucleotide that is complementary to a polynucleotide in (a),(b), (c), (d), (e), (f) or (g).

According to one aspect, the present invention provides an isolatedpolynucleotide comprising a polynucleotide chosen from:

(a) a polynucleotide encoding a polypeptide having at least 70% identityto a second polypeptide comprising a sequence chosen from: SEQ ID NO: 2,4, 6, 8, 10, 12 or 14;

(b) a polynucleotide encoding a polypeptide having at least 80% identityto a second polypeptide comprising a sequence chosen from: SEQ ID NO: 2,4, 6, 8, 10, 12 or 14;

(c) a polynucleotide encoding a polypeptide having at least 95% identityto a second polypeptide comprising a sequence chosen from: SEQ ID NO: 2,4, 6, 8, 10, 12 or 14;

(d) a polynucleotide encoding a polypeptide comprising a sequence chosenfrom: SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14;

(e) a polynucleotide encoding a polypeptide capable of raisingantibodies having binding specificity for a polypeptide comprising asequence chosen from: SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14;

(f) a polynucleotide encoding an epitope bearing portion of apolypeptide comprising a sequence chosen from SEQ ID NO: 2, 4, 6, 8, 10,12 or 14;

(g) a polynucleotide comprising a sequence chosen from SEQ ID NO: 1, 3,5, 7, 9, 11 and 13;

(h) a polynucleotide that is complementary to a polynucleotide in (a),(b), (c), (d), (e), (f) or (g).

According to one aspect, the present invention provides an isolatedpolypeptide comprising a polypeptide chosen from:

(a) a polypeptide having at least 70% identity to a second polypeptidecomprising a sequence chosen from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 orfragments or analogs thereof;

(b) a polypeptide having at least 80% identity to a second polypeptidecomprising a sequence chosen from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 orfragments or analogs thereof;

(c) a polypeptide having at least 95% identity to a second polypeptidecomprising a sequence chosen from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 orfragments or analogs thereof;

(d) a polypeptide comprising a sequence chosen from SEQ ID NO: 2, 4, 6,8, 10, 12, 14 or fragments or analogs thereof;

(e) a polypeptide capable of raising antibodies having bindingspecificity for a polypeptide comprising a sequence chosen from SEQ IDNO: 2, 4, 6, 8, 10, 12, 14 or fragments or analogs thereof;

(f) an epitope bearing portion of a polypeptide comprising a sequencechosen from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or fragments or analogsthereof;

(g) the polypeptide of (a), (b), (c), (d), (e) or (f) wherein theN-terminal Met residue is deleted;

(h) the polypeptide of (a), (b), (c), (d), (e) or (f) wherein thesecretory amino acid sequence is deleted.

According to one aspect, the present invention provides an isolatedpolypeptide comprising a polypeptide chosen from:

(a) a polypeptide having at least 70% identity to a second polypeptidecomprising a sequence chosen from SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14;

(b) a polypeptide having at least 80% identity to a second polypeptidecomprising a sequence chosen from SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14;

(c) a polypeptide having at least 95% identity to a second polypeptidecomprising a sequence chosen from SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14;

(d) a polypeptide comprising a sequence chosen from SEQ ID NO: 2, 4, 6,8, 10, 12 or 14;

(e) a polypeptide capable of raising antibodies having bindingspecificity for a polypeptide comprising a sequence chosen from SEQ IDNO: 2, 4, 6, 8, 10, 12 or 14;

(f) an epitope bearing portion of a polypeptide comprising a sequencechosen from SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14;

(g) the polypeptide of (a), (b), (c), (d), (e) or (f) wherein theN-terminal Met residue is deleted;

(h) the polypeptide of (a), (b), (c), (d), (e) or (f) wherein thesecretory amino acid sequence is deleted.

Those skilled in the art will appreciate that the invention includes DNAmolecules, i.e. polynucleotides and their complementary sequences thatencode analogs such as mutants, variants, homologues and derivatives ofsuch polypeptides, as described herein in the present patentapplication. The invention also includes RNA molecules corresponding tothe DNA molecules of the invention. In addition to the DNA and RNAmolecules, the invention includes the corresponding polypeptides andmonospecific antibodies that specifically bind to such polypeptides.

In a further embodiment, the polypeptides in accordance with the presentinvention are antigenic.

In a further embodiment, the polypeptides in accordance with the presentinvention are immunogenic.

In a further embodiment, the polypeptides in accordance with the presentinvention can elicit an immune response in a host.

In a further embodiment, the present invention also relates topolypeptides which are able to raise antibodies having bindingspecificity to the polypeptides of the present invention as definedabove.

An antibody that “has binding specificity” is an antibody thatrecognizes and binds the selected polypeptide but which does notsubstantially recognize and bind other molecules in a sample, e.g., abiological sample, which naturally includes the selected peptide.Specific binding can be measured using an ELISA assay in which theselected polypeptide is used as an antigen.

In accordance with the present invention, “protection” in the biologicalstudies is defined by a significant increase in the survival curve, rateor period. Statistical analysis using the Log rank test to comparesurvival curves, and Fisher exact test to compare survival rates andnumbers of days to death, respectively, might be useful to calculate Pvalues and determine whether the difference between the two groups isstatistically significant. P values of 0.05 are regarded as notsignificant.

In an additional aspect of the invention there are providedantigenic/immunogenic fragments of the polypeptides of the invention, orof analogs thereof.

The fragments of the present invention should include one or more suchepitopic regions or be sufficiently similar to such regions to retaintheir antigenic/immunogenic properties. Thus, for fragments according tothe present invention the degree of identity is perhaps irrelevant,since they may be 100% identical to a particular part of a polypeptideor analog thereof as described herein. The present invention furtherprovides fragments having at least 10 contiguous amino acid residuesfrom the polypeptide sequences of the present invention. In oneembodiment, at least 15 contiguous amino acid residues. In oneembodiment, at least 20 contiguous amino acid residues.

The skilled person will appreciate that analogs of the polypeptides ofthe invention will also find use in the context of the presentinvention, i.e. as antigenic/immunogenic material. Thus, for instanceproteins or polypeptides which include one or more additions, deletions,substitutions or the like are encompassed by the present invention.

As used herein, “fragments”, “analogs” or “derivatives” of thepolypeptides of the invention include those polypeptides in which one ormore of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably conserved) and which may benatural or unnatural. In one embodiment, derivatives and analogs ofpolypeptides of the invention will have about 80% identity with thosesequences illustrated in the figures or fragments thereof. That is, 80%of the residues are the same. In a further embodiment, polypeptides willhave greater than 80% identity. In a further embodiment, polypeptideswill have greater than 85% identity. In a further embodiment,polypeptides will have greater than 90% identity. In a furtherembodiment, polypeptides will have greater than 95% identity. In afurther embodiment, polypeptides will have greater than 99% identity. Ina further embodiment, analogs of polypeptides of the invention will havefewer than about 20 amino acid residue substitutions, modifications ordeletions and more preferably less than 10.

These substitutions are those having a minimal influence on thesecondary structure and hydropathic nature of the polypeptide. Preferredsubstitutions are those known in the art as conserved, i.e. thesubstituted residues share physical or chemical properties such ashydrophobicity, size, charge or functional groups. These includesubstitutions such as those described by Dayhoff, M. in Atlas of ProteinSequence and Structure 5, 1978 and by Argos, P. in EMBO J. 8, 779-785,1989. For example, amino acids, either natural or unnatural, belongingto one of the following groups represent conservative changes:

ala, pro, gly, gln, asn, ser, thr, val; cys, ser, tyr, thr; val, ile,leu, met, ala, phe; lys, arg, orn, his; and phe, tyr, trp, his.The preferred substitutions also include substitutions of D-enantiomersfor the corresponding L-amino acids.

In an alternative approach, the analogs could be fusion polypeptides,incorporating moieties which render purification easier, for example byeffectively tagging the desired polypeptide. It may be necessary toremove the “tag” or it may be the case that the fusion polypeptideitself retains sufficient antigenicity to be useful.

The percentage of homology is defined as the sum of the percentage ofidentity plus the percentage of similarity or conservation of amino acidtype.

In one embodiment, analogs of polypeptides of the invention will haveabout 70% identity with those sequences illustrated in the figures orfragments thereof. That is, 70% of the residues are the same. In afurther embodiment, polypeptides will have greater than 80% identity. Ina further embodiment, polypeptides will have greater than 85% identity.In a further embodiment, polypeptides will have greater than 90%identity. In a further embodiment, polypeptides will have greater than95% identity. In a further embodiment, polypeptides will have greaterthan 99% identity. In a further embodiment, analogs of polypeptides ofthe invention will have fewer than about 20 amino acid residuesubstitutions, modifications or deletions and more preferably less than10.

In one embodiment, analogs of polypeptides of the invention will haveabout 70% homology with those sequences illustrated in the figures orfragments thereof. In a further embodiment, polypeptides will havegreater than 80% homology. In a further embodiment, polypeptides willhave greater than 85% homology. In a further embodiment, polypeptideswill have greater than 90% homology. In a further embodiment,polypeptides will have greater than 95% homology. In a furtherembodiment, polypeptides will have greater than 99% homology. In afurther embodiment, analogs of polypeptides of the invention will havefewer than about 20 amino acid residue substitutions, modifications ordeletions and more preferably less than 10.

One can use a program such as the CLUSTAL program to compare amino acidsequences. This program compares amino acid sequences and finds theoptimal alignment by inserting spaces in either sequence as appropriate.It is possible to calculate amino acid identity or homology for anoptimal alignment. A program like BLASTx will align the longest stretchof similar sequences and assign a value to the fit. It is thus possibleto obtain a comparison where several regions of similarity are found,each having a different score. Both types of identity analysis arecontemplated in the present invention.

In an alternative approach, the analogs or derivatives could be fusionpolypeptides, incorporating moieties which render purification easier,for example by effectively tagging the desired protein or polypeptide,it may be necessary to remove the “tag” or it may be the case that thefusion polypeptide itself retains sufficient antigenicity to be useful.

It is well known that it is possible to screen an antigenic polypeptideto identify epitopic regions, i.e. those regions which are responsiblefor the polypeptide's antigenicity or immunogenicity. Methods forcarrying out such screening are well known in the art. Thus, thefragments of the present invention should include one or more suchepitopic regions or be sufficiently similar to such regions to retaintheir antigenic/immunogenic properties.

Thus, for fragments according to the present invention the degree ofidentity is perhaps irrelevant, since they may be 100% identical to aparticular part of a polypeptide, analog as described herein.

Thus, what is important for analogs, derivatives and fragments is thatthey possess at least a degree of the antigenicity/immunogenicity of theprotein or polypeptide from which they are derived.

Also included are polypeptides which have fused thereto other compoundswhich alter the polypeptides biological or pharmacological propertiesi.e. polyethylene glycol (PEG) to increase half-life; leader orsecretory amino acid sequences for ease of purification; prepro- andpro-sequences; and (poly)saccharides.

Furthermore, in those situations where amino acid regions are found tobe polymorphic, it may be desirable to vary one or more particular aminoacids to more effectively mimic the different epitopes of the differentMoraxella strains.

Moreover, the polypeptides of the present invention can be modified byterminal —NH₂ acylation (eg. by acetylation, or thioglycolic acidamidation, terminal carboxy amidation, e.g. with ammonia or methylamine)to provide stability, increased hydrophobicity for linking or binding toa support or other molecule.

Also contemplated are hetero and homo polypeptide multimers of thepolypeptide fragments and analogues. These polymeric forms include, forexample, one or more polypeptides that have been cross-linked withcross-linkers such as avidin/biotin, gluteraldehyde ordimethyl-superimidate. Such polymeric forms also include polypeptidescontaining two or more tandem or inverted contiguous sequences, producedfrom multicistronic mRNAs generated by recombinant DNA technology.

In a further embodiment, the present invention also relates to chimericpolypeptides which comprise one or more polypeptides or fragments oranalogs thereof as defined in the figures of the present application.

In a further embodiment, the present invention also relates to chimericpolypeptides comprising two or more polypeptides having a sequencechosen from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or fragments or analogsthereof; provided that the polypeptides are linked as to formed achimeric polypeptide.

In a further embodiment, the present invention also relates to chimericpolypeptides comprising two or more polypeptides comprising a sequencechosen from SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14 provided that thepolypeptides are linked as to formed a chimeric polypeptide.

Preferably, a fragment, analog or derivative of a polypeptide of theinvention will comprise at least one antigenic region i.e. at least oneepitope.

In order to achieve the formation of antigenic polymers (i.e. syntheticmultimers), polypeptides may be utilized having bishaloacetyl groups,nitroarylhalides, or the like, where the reagents being specific forthio groups. Therefore, the link between two mercapto groups of thedifferent polypeptides may be a single bond or may be composed of alinking group of at least two, typically at least four, and not morethan 16, but usually not more than about 14 carbon atoms.

In a particular embodiment, polypeptide fragments and analogs of theinvention do not contain a starting residue, such as methionine (Met) orvaline (Val). Preferably, polypeptides will not incorporate a leader orsecretory sequence (signal sequence). The signal portion of apolypeptide of the invention may be determined according to establishedmolecular biological techniques. In general, the polypeptide of interestmay be isolated from a Moraxella culture and subsequently sequenced todetermine the initial residue of the mature protein and therefore thesequence of the mature polypeptide.

It is understood that polypeptides can be produced and/or used withouttheir start codon (methionine or valine) and/or without their leaderpeptide to favor production and purification of recombinantpolypeptides. It is known that cloning genes without sequences encodingleader peptides will restrict the polypeptides to the cytoplasm of E.coli and will facilitate their recovery (Glick, B. R. and Pasternak, J.J. (1998) Manipulation of gene expression in prokaryotes. In “Molecularbiotechnology: Principles and applications of recombinant DNA”, 2ndedition, ASM Press, Washington D.C., p.109-143).

According to another aspect of the invention, there are also provided(i) a composition of matter containing a polypeptide of the invention,together with a carrier, diluent or adjuvant; (ii) a pharmaceuticalcomposition comprising a polypeptide of the invention and a carrier,diluent or adjuvant; (iii) a vaccine comprising a polypeptide of theinvention and a carrier, diluent or adjuvant; (iv) a method for inducingan immune response against Moraxella, in a host, by administering to thehost, an immunogenically effective amount of a polypeptide of theinvention to elicit an immune response, e.g., a protective immuneresponse to Moraxella; and particularly, (v) a method for preventingand/or treating a Moraxella infection, by administering a prophylacticor therapeutic amount of a polypeptide of the invention to a host inneed.

According to another aspect of the invention, there are also provided(i) a composition of matter containing a polynucleotide of theinvention, together with a carrier, diluent or adjuvant; (ii) apharmaceutical composition comprising a polynucleotide of the inventionand a carrier, diluent or adjuvant; (iii) a method for inducing animmune response against Moraxella, in a host, by administering to thehost, an immunogenically effective amount of a polynucleotide of theinvention to elicit an immune response, e.g., a protective immuneresponse to Moraxella; and particularly, (iv) a method for preventingand/or treating a Moraxella infection, by administering a prophylacticor therapeutic amount of a polynucleotide of the invention to a host inneed. Before immunization, the polypeptides of the invention can also becoupled or conjugated to carrier proteins such as tetanus toxin,diphtheria toxin, hepatitis B virus surface antigen, poliomyelitis virusVP1 antigen or any other viral or bacterial toxin or antigen or anysuitable proteins to stimulate the development of a stronger immuneresponse. This coupling or conjugation can be done chemically orgenetically. A more detailed description of peptide-carrier conjugationis available in Van Regenmortel, M. H. V., Briand J. P., Muller S.,Plaué S., <<Synthetic Polypeptides as antigens>> in LaboratoryTechniques in Biochemistry and Molecular Biology, Vol.19 (ed.) Burdou,R. H. & Van Knippenberg P. H. (1988), Elsevier New York.

According to another aspect, there are provided pharmaceuticalcompositions comprising one or more Moraxella polypeptides of theinvention in a mixture with a pharmaceutically acceptable adjuvant.Suitable adjuvants include (1) oil-in-water emulsion formulations suchas MF59™, SAF™, Ribi™; (2) Freund's complete or incomplete adjuvant; (3)salts i.e. AlK(SO₄)₂, AlNa(SO₄)₂, AlNH₄(SO₄)₂, Al(OH)₃, AlPO₄, silica,kaolin; (4) saponin derivatives such as Stimulon™ or particles generatedtherefrom such as ISCOMs (immunostimulating complexes); (5) cytokinessuch as interleukins, interferons, macrophage colony stimulating factor(M-CSF), tumor necrosis factor (TNF); (6) other substances such ascarbon polynucleotides i.e. poly IC and poly AU, detoxified choleratoxin (CTB) and E. coli heat labile toxin for induction of mucosalimmunity. A more detailed description of adjuvant is available in areview by M. Z. I Khan et al. in Pharmaceutical Research, vol. 11, No. 1(1994) pp2-11, and also in another review by Gupta et al., in Vaccine,Vol. 13, No. 14, pp1263-1276 (1995) and in WO 99/24578. Preferredadjuvants include QuilA™, QS21™, Alhydrogel™ and Adjuphos™.

Pharmaceutical compositions of the invention may be administeredparenterally by injection, rapid infusion, nasopharyngeal absorption,dermoabsorption, or buccal or oral.

Pharmaceutical compositions of the invention are used for theprophylaxis of Moraxella infection and/or diseases and symptoms mediatedby Moraxella infection as described in Manual of Clinical Microbiology,P. R. Murray (Ed, in chief), E. J. Baron, M. A. Pfaller, F. C. Tenoverand R. H. Yolken. ASM Press, Washington, D.C. seventh edition, 1999,1773p. In one embodiment, pharmaceutical compositions of the presentinvention are used for the treatment or prophylaxis of otitis media,sinusitis, persistent cough, acute laryngitis, suppurative keratitis,conjunctivitis neonatorum. In one embodiment, vaccine compositions ofthe invention are used for the treatment or prophylaxis of Moraxellainfection and/or diseases and symptoms mediated by Moraxella infection.In a further embodiment, the Moraxella infection is Moraxellacatarrhalis.

In a further embodiment, the invention provides a method for prophylaxisor treatment of Moraxella infection in a host susceptible to Moraxellainfection comprising administering to said host a prophylactic ortherapeutic amount of a composition of the invention.

As used in the present application, the term “host” includes mammals. Ina further embodiment, the mammal is human.

In a particular embodiment, pharmaceutical compositions are administeredto those hosts at risk of Moraxella infection such as neonates, infants,children, elderly and immunocompromised hosts.

In a particular embodiment, pharmaceutical compositions are administeredto those hosts at risk of Moraxella infection such as adults.

Pharmaceutical compositions are preferably in unit dosage form of about0.001 to 100 μg/kg (antigen/body weight) and more preferably 0.01 to 10μg/kg and most preferably 0.1 to 1 μg/kg 1 to 3 times with an intervalof about 1 to 6 week intervals between immunizations.

Pharmaceutical compositions are preferably in unit dosage form of about0.1 μg to 10 mg and more preferably 1 g to 1 mg and most preferably 10to 100 μg 1 to 3 times with an interval of about 1 to 6 week intervalsbetween immunizations.

According to another aspect, there are provided polynucleotides encodingpolypeptides characterized by the amino acid sequence comprising asequence chosen from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or fragments oranalogs thereof.

In one embodiment, polynucleotides are those illustrated in SEQ ID No:1, 3, 5, 7, 9, 11, 13 which may include the open reading frames (ORF),encoding the polypeptides of the invention.

It will be appreciated that the polynucleotide sequences illustrated inthe figures may be altered with degenerate codons yet still encode thepolypeptides of the invention. Accordingly the present invention furtherprovides polynucleotides which hybridize to the polynucleotide sequencesherein above described (or the complement sequences thereof) having 70%identity between sequences. In one embodiment, at least 80% identitybetween sequences. In one embodiment, at least 85% identity betweensequences. In one embodiment, at least 90% identity between sequences.In a further embodiment, polynucleotides are hybridizable understringent conditions i.e. having at least 95% identity. In a furtherembodiment, more than 97% identity.

Suitable stringent conditions for hybridation can be readily determinedby one of skilled in the art (see for example Sambrook et al., (1989)Molecular cloning: A Laboratory Manual, 2^(nd) ed, Cold Spring Harbor,N.Y.; Current Protocols in Molecular Biology, (1999) Edited by AusubelF. M. et al., John Wiley & Sons, Inc., N.Y.).

In a further embodiment, the present invention provides polynucleotidesthat hybridize under stringent conditions to either

(a) a DNA sequence encoding a polypeptide or

(b) the complement of a DNA sequence encoding a polypeptide;

wherein said polypeptide comprises a sequence chosen from SEQ ID NO: 2,4, 6, 8, 10, 12 or 14 or fragments or analogs thereof.

In a further embodiment, the present invention provides polynucleotidesthat hybridize under stringent conditions to either

(a) a DNA sequence encoding a polypeptide or

(b) the complement of a DNA sequence encoding a polypeptide;

wherein said polypeptide comprises a sequence chosen from SEQ ID NO: 2,4, 6, 8, 10, 12 or 14.

In a further embodiment, the present invention provides polynucleotidesthat hybridize under stringent conditions to either

(a) a DNA sequence encoding a polypeptide or

(b) the complement of a DNA sequence encoding a polypeptide;

wherein said polypeptide comprises at least 10 contiguous amino acidresidues from a polypeptide comprising a sequence chosen from SEQ ID NO:2, 4, 6, 8, 10, 12 or 14 or fragments or analogs thereof.

In a further embodiment, the present invention provides polynucleotidesthat hybridize under stringent conditions to either

(a) a DNA sequence encoding a polypeptide or

(b) the complement of a DNA sequence encoding a polypeptide;

wherein said polypeptide comprises at least 10 contiguous amino acidresidues from a polypeptide comprising a sequence chosen from SEQ ID NO:2, 4, 6, 8, 10, 12 or 14.

In a further embodiment, polynucleotides are those illustrated in SEQ IDNO: 1, 3, 5, 7, 9, 11, 13 or fragments or analogs thereof encodingpolypeptides of the invention.

In a further embodiment, polynucleotides are those illustrated in SEQ IDNO: 1, 3, 5, 7, 9, 11 or 13 encoding polypeptides of the invention.

As will be readily appreciated by one skilled in the art,polynucleotides include both DNA and RNA.

The present invention also includes polynucleotides complementary to thepolynucleotides described in the present application.

In a further aspect, polynucleotides encoding polypeptides of theinvention, or fragments, analogs or derivatives thereof, may be used ina DNA immunization method. That is, they can be incorporated into avector which is replicable and expressible upon injection therebyproducing the antigenic polypeptide in vivo. For example polynucleotidesmay be incorporated into a plasmid vector under the control of the CMVpromoter which is functional in eukaryotic cells. Preferably the vectoris injected intramuscularly.

According to another aspect, there is provided a process for producingpolypeptides of the invention by recombinant techniques by expressing apolynucleotide encoding said polypeptide in a host cell and recoveringthe expressed polypeptide product. Alternatively, the polypeptides canbe produced according to established synthetic chemical techniques i.e.solution phase or solid phase synthesis of oligopeptides which areligated to produce the full polypeptide (block ligation)

General methods for obtention and evaluation of polynucleotides andpolypeptides are described in the following references: Sambrook et al,Molecular Cloning: A Laboratory Manual, 2nd ed, Cold Spring Harbor,N.Y., 1989; Current Protocols in Molecular Biology, Edited by Ausubel F.M. et al., John Wiley and Sons, Inc. New York; PCR Cloning Protocols,from Molecular Cloning to Genetic Engineering, Edited by White B. A.,Humana Press, Totowa, N.J., 1997, 490 pages; Protein Purification,Principles and Practices, Scopes R. K.; Springer-verlag, New York, 3rdEdition, 1993, 380 pages; Current Protocols in Immunology, Edited byColigan J. E. et al., John Wiley & Sons Inc., New York.

The present invention provides a process for producing a polypeptidecomprising culturing a host cell of the invention under conditionssuitable for expression of said polypeptide.

For recombinant production, host cells are transfected with vectorswhich encode the polypeptides of the invention, and then cultured in anutrient media modified as appropriate for activating promoters,selecting transformants or amplifying the genes. Suitable vectors arethose that are viable and replicable in the chosen host and includechromosomal, non-chromosomal and synthetic DNA sequences e.g. bacterialplasmids, phage DNA, baculovirus, yeast plasmids, vectors derived fromcombinations of plasmids and phage DNA. The polypeptide sequence may beincorporated in the vector at the appropriate site using restrictionenzymes such that it is operably linked to an expression control regioncomprising a promoter, ribosome binding site (consensus region orShine-Dalgarno sequence), and optionally an operator (control element).One can select individual components of the expression control regionthat are appropriate for a given host and vector according toestablished molecular biology principles (Sambrook et al, MolecularCloning: A Laboratory Manual, 2nd ed, Cold Spring Harbor, N.Y., 1989;Current Protocols in Molecular Biology, Edited by Ausubel F. M. et al.,John Wiley and Sons, Inc. New York). Suitable promoters include but arenot limited to LTR or SV40 promoter, E. coli lac, tac or trp promotersand the phage lambda PL promoter. Vectors will preferably incorporate anorigin of replication as well as selection markers i.e. ampicilinresistance gene. Suitable bacterial vectors include pET, pQE70, pQE60,pQE-9, pD10 phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a,pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 andeukaryotic vectors pBlueBacIII, pWLNEO, pSV2CAT, pOG44, pXT1, pSG,pSVK3, pBPV, pMSG and pSVL. Host cells may be bacterial i.e. E. coli,Bacillus subtilis, Streptomyces; fungal i.e. Aspergillus niger,Aspergillus nidulins; yeast i.e. Saccharomyces or eukaryotic i.e. CHO,COS.

Upon expression of the polypeptide in culture, cells are typicallyharvested by centrifugation then disrupted by physical or chemical means(if the expressed polypeptide is not secreted into the media) and theresulting crude extract retained to isolate the polypeptide of interest.Purification of the polypeptide from culture media or lysate may beachieved by established techniques depending on the properties of thepolypeptide i.e. using ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, hydroxylapatitechromatography and lectin chromatography. Final purification may beachieved using HPLC.

The polypeptides may be expressed with or without a leader or secretionsequence. In the former case the leader may be removed usingpost-translational processing (see U.S. Pat. No. 4,431,739; U.S. Pat.No. 4,425,437; and U.S. Pat. No. 4,338,397) or be chemically removedsubsequent to purifying the expressed polypeptide.

According to a further aspect, the Moraxella polypeptides of theinvention may be used in a diagnostic test for Moraxella infection, inparticular Moraxella catarrhalis infection. Several diagnostic methodsare possible, for example detecting Moraxella organism in a biologicalsample, the following procedure may be followed:

a) obtaining a biological sample from a host;

b) incubating an antibody or fragment thereof reactive with a Moraxellapolypeptide of the invention with the biological sample to form amixture; and

c) detecting specifically bound antibody or bound fragment in themixture which indicates the presence of Moraxella.

Alternatively, a method for the detection of antibody specific to aMoraxella antigen in a biological sample containing or suspected ofcontaining said antibody may be performed as follows:

a) obtaining a biological sample from a host;

b) incubating one or more Moraxella polypeptides of the invention orfragments thereof with the biological sample to form a mixture; and

c) detecting specifically bound antigen or bound fragment in the mixturewhich indicates the presence of antibody specific to Moraxella.

One of skill in the art will recognize that this diagnostic test maytake several forms, including an immunological test such as anenzyme-linked immunosorbent assay (ELISA), a radioimmunoassay or a latexagglutination assay, essentially to determine whether antibodiesspecific for the protein are present in an organism.

The DNA sequences encoding polypeptides of the invention may also beused to design DNA probes for use in detecting the presence of Moraxellain a biological sample suspected of containing such bacteria. Thedetection method of this invention comprises:

a) obtaining the biological sample from a host;

b) incubating one or more DNA probes having a DNA sequence encoding apolypeptide of the invention or fragments thereof with the biologicalsample to form a mixture; and

c) detecting specifically bound DNA probe in the mixture which indicatesthe presence of Moraxella bacteria.

The DNA probes of this invention may also be used for detectingcirculating Moraxella i.e. Moraxella nucleic acids in a sample, forexample using a polymerase chain reaction, as a method of diagnosingMoraxella infections. The probe may be synthesized using conventionaltechniques and may be immobilized on a solid phase, or may be labelledwith a detectable label. A preferred DNA probe for this application isan oligomer having a sequence complementary to at least about 6contiguous nucleotides of the Moraxella polypeptides of the invention.In a further embodiment, the preferred DNA probe will be an oligomerhaving a sequence complementary to at least about 15 contiguousnucleotides of the Moraxella polypeptides of the invention. In a furtherembodiment, the preferred DNA probe will be an oligomer having asequence complementary to at least about 30 contiguous nucleotides ofthe Moraxella polypeptides of the invention. In a further embodiment,the preferred DNA probe will be an oligomer having a sequencecomplementary to at least about 50 contiguous nucleotides of theMoraxella polypeptides of the invention.

Another diagnostic method for the detection of Moraxella in a hostcomprises:

a) labelling an antibody reactive with a polypeptide of the invention orfragment thereof with a detectable label;

b) administering the labelled antibody or labelled fragment to the host;and

c) detecting specifically bound labelled antibody or labelled fragmentin the host which indicates the presence of Moraxella.

Alternatively, a method for the detection of antibody specific to aMoraxella antigen in a biological sample containing or suspected ofcontaining said antibody may be performed as follows:

a) obtaining a biological sample from a host;

b) incubating one or more Moraxella polypeptides of the invention orfragments thereof with the biological sample to form a mixture; and

c) detecting specifically bound antigen or bound fragment in the mixturewhich indicates the presence of antibody specific to Moraxella.

One of skill in the art will recognize that the diagnostic test may takeseveral forms, including an immunological test such as an enzyme-linkedimmunosorbent assay (ELISA), a radioimmunoassay or a latex agglutinationassay, essentially to determine whether antibodies specific for theprotein are present in an organism.

The DNA sequences encoding polypeptides of the invention may also beused to design DNA probes for use in detecting the presence of Moraxellain a biological sample suspected of containing such bacteria. Thedetection method of this invention comprises:

a) obtaining the biological sample from a host;

b) incubating one or more DNA probes having a DNA sequence encoding apolypeptide of the invention or fragments thereof with the biologicalsample to form a mixture; and

c) detecting specifically bound DNA probe in the mixture which indicatesthe presence of Moraxella bacteria.

According to one aspect, the present invention provides the use of anantibody for treatment and/or prophylaxis of Moraxella infections.

A further aspect of the invention is the use of the Moraxellapolypeptides of the invention as immunogens for the production ofspecific antibodies for the diagnosis and in particular the treatment ofMoraxella infection. Suitable antibodies may be determined usingappropriate screening methods, for example by measuring the ability of aparticular antibody to passively protect against Moraxella infection ina test model. One example of an animal model is the mouse modeldescribed in the examples herein. The antibody may be a whole antibodyor an antigen-binding fragment thereof and may belong to anyimmunoglobulin class. The antibody or fragment may be of animal origin,specifically of mammalian origin and more specifically of murine, rat orhuman origin. It may be a natural antibody or a fragment thereof, or ifdesired, a recombinant antibody or antibody fragment. The termrecombinant antibody or antibody fragment means antibody or antibodyfragment which was produced using molecular biology techniques. Theantibody or antibody fragments may be polyclonal, or preferablymonoclonal. It may be specific for a number of epitopes associated withthe Moraxella polypeptides but is preferably specific for one.

According to one aspect, the present invention provides the use of anantibody for prophylaxis and/or treatment of Moraxella infections.

A further aspect of the invention is the use of the Moraxellapolypeptides of the invention as immunogens for the production ofspecific antibodies for the diagnosis and in particular the treatment ofMoraxella infection. Suitable antibodies may be determined usingappropriate screening methods, for example by measuring the ability of aparticular antibody to passively protect against Moraxella infection ina test model. One example of an animal model is the mouse modeldescribed in the examples herein. The antibody may be a whole antibodyor an antigen-binding fragment thereof and may belong to anyimmunoglobulin class. The antibody or fragment may be of animal origin,specifically of mammalian origin and more specifically of murine, rat orhuman origin. It may be a natural antibody or a fragment thereof, or ifdesired, a recombinant antibody or antibody fragment. The termrecombinant antibody or antibody fragment means antibody or antibodyfragment which was produced using molecular biology techniques. Theantibody or antibody fragments may be polyclonal, or preferablymonoclonal. It may be specific for a number of epitopes associated withthe Moraxella polypeptides but is preferably specific for one.

A further aspect of the invention is the use of the antibodies directedto the polypeptides of the invention for passive immunization. One coulduse the antibodies described in the present application.

A further aspect of the invention is a method for immunization, wherebyan antibody raised by a polypeptide of the invention is administered toa host in an amount sufficient to provide a passive immunization.

In a further embodiment, the invention provides the use of apharmaceutical composition of the invention in the manufacture of amedicament for the prophylactic or therapeutic treatment of Moraxellainfection.

In a further embodiment, the invention provides a kit comprising apolypeptide of the invention for detection or diagnosis of Moraxellainfection.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

EXAMPLE 1

This example illustrates the cloning and molecular characteristics ofBVH-MC2 gene and corresponding polypeptide.

The coding region of M. catarrhalis BVH-MC2 (SEQ ID NO: 1) gene wasamplified by PCR (DNA Thermal Cycler GeneAmp® PCR system 2400 PerkinElmer, San Jose, Calif.) from genomic DNA of M. catarrhalis strain ETSUC-2 using the following oligos that contained base extensions for theaddition of restriction sites NdeI (CATATG) and XhoI (CTCGAG): DMAR544(5′-CATCAGTGCATATGAATACGACACACCATCACACG-3′) (SEQ ID NO:15); DMAR545(5′-GAGTTATTCTCGAGTTTGTCCAAATTTGGCTTAGTTTTAC-3′) (SEQ ID NO:16). PCRproducts were purified from agarose gel using a QIA® quick gelextraction kit from QIAgen® following the manufacturer's instructions(Chatsworth, Calif.), and digested with NdeI and XhoI (AmershamPharmacia Biotech, Inc, Baie d'Urfė, Canada). The pET21b(+) vector(Novagen, Madison, Wis.) was digested with NdeI and XhoI and purifiedfrom agarose gel using a QIA® quick gel extraction kit from QIAgen®(Chatsworth, Calif.). The NdeI-XhoI PCR products were ligated to theNdeI-XhoI pET21b(+) expression vector. The ligated products weretransformed into E. coli strain DH5α [φ80dlacZΔM15 Δ(lacZYA-argF)U169endA1 recA1 hsdR17(r_(K)−m_(K)+) deoR thi-1 supE44 λ⁻gyrA96 relA1](Gibco BRL, Gaithersburg, Md.) according to the method of Simanis(Hanahan, D. DNA Cloning, 1985, D.M. Glover (ed), pp. 109-135).Recombinant pET21b(+) plasmid (rpET21b(+)) containing BVH-MC2 gene waspurified using a QIAgen® kit (Chatsworth, Calif.) and DNA insert wassequenced (Taq Dye Deoxy Terminator Cycle Sequencing kit, ABI, FosterCity, Calif.).

TABLE 1 Oligonucleotide primers used for PCR amplification of M.catarrhalis genes. Primers Restriction Genes I.D. site Vector SequenceBVH-MC2 DMAR544 NdeI pET21b 5′-CATCAGTGCATATGAATACGACACACCATCACACG-3′(+) (SEQ ID No: 15) BVH-MC2 DMAR545 XhoI pET21b5′-GAGTTATTCTCGAGTTTGTCCAAATTTGGCTTAGTTTTAC-3′ (+) (SEQ ID No: 16)BVH-MC2 DMAR544a BglII pCMV-GH 5′-TCAGTGAGATCTTGAATACGACACACCATC-3′ (SEQID No: 17) BVH-MC2 DMAR545a SalI pCMV-GH5′-GATTTGAGTTGTCGACTTATTTGTCCAAATTTG-3′ (SEQ ID No: 18) BVH-MC3 DMAR592NdeI pET21b 5′-CGGAGTGCCATATGAGCTTAATTAATAAATTAAATG-3′ (+) (SEQ ID No:19) BVH-MC3 BMAR593 XhoI pET21b 5′-TATAACTCGAGGTTTTGTGCAACAGGTGTTG-3′(+) (SEQ ID No: 20) BVH-MC3 DMAR592a BglII pCMV-GH5′-CGCTTGAGATCTTGGAAGATGTGTATCAGCGTGC-3′ (SEQ ID No: 21) BVH-MC3DMAR593a HindIII pCMV-GH 5′-CAATAACAAAGCTTTCAGTTTTGTGCAACAGGTGTTG-3′(SEQ ID No: 22) BVH-MC4 RIOS71 NdeI pET21b5′-AACCGCACATATGTATCGCTTGGTGTCACCACC-3′ (+) (SEQ ID No: 23) BVH-MC4RIOS72 XhoI pET21b 5′-GGTGACTCGAGGTACTCATCACCAACTAATCGCAC-3′ (+) (SEQ IDNo: 24) BVH-MC4 RIOS71a BamHI pCMV-GH 5′-GCAGGATCCTTATCGCTTGGTGTCACC-3′(SEQ ID No: 25) BVH-MC4 RIOS72a SalI pCMV-GH5′-ATCAATCGGGTCGACTTAGTACTCATCACCA-3′ (SEQ ID No: 26) BVH-MC5 RIOS59NdeI pET21b 5′-AAAGCTTCATATGGCCCAAAGTCAAGAATCTGCC-3′ (+) (SEQ ID No: 27)BVH-MC5 RIOS60 XhoI pET21b 5′-CGATAACTCGAGTTGAACATCAGGCACCTGC-3′ (+)(SEQ ID No: 28) BVH-MC5 RIOS59a BglII pCMV-GH5′-ACCATTCAAAAGAGATCTTGGCCCAAAGTCAAGAATCTG-3′ (SEQ ID No: 29) BVH-MC5RIOS60a SalI pCMV-GH 5′-GTTAGACCGAGTCGACTCATTGAACATCAGGCA-3′ (SEQ ID No:30)

It was determined that the open reading frame (ORF) which codes forBVH-MC2 polypeptide contains 1467-bp and encodes a 488 amino acidresidues polypeptide with a predicted pI of 6.08 and a predictedmolecular mass of 53754.35 Da. Analysis of the predicted amino acidresidues sequence (SEQ ID NO: 2) using the Spscan software (WisconsinSequence Analysis Package; Genetics Computer Group) suggested theexistence of a 30 amino acid residues signal peptide(MDTDMKHLTKHRLSAAIIGVLLFISPSVQA) (SEQ ID NO:31), which ends with acleavage site located between an alanine and an asparagine residues.

To confirm the presence by PCR amplification of BVH-MC2 (SEQ ID NO: 1)gene, the following 4 distinct M. catarrhalis strains were used: M.catarrhalis ETSU C-2, ETSU T-25, and ETSU 658 clinical isolates wereprovided by the East Tennessee State University; M. catarrhalis strainM-12 was provided by the centre de recherche en infectiologie du centrehospitalier de l'université Laval. The E. coli XL1-Blue MRF′ was used inthese experiments as negative control. BVH-MC2 (SEQ ID NO: 1) gene wasamplified by PCR (DNA Thermal Cycler GeneAmp PCR system 2400 PerkinElmer, San Jose, Calif.) from genomic DNA from the 4 M. catarrhalisstrains, and the control E. coli strain using the oligonucleotidesprimers DMAR544 and DMAR545 (Table 1). PCR was performed with 30 cyclesof 30 sec at 94° C., 30 sec at 51° C. and 1 min 20 sec at 72° C. and afinal elongation period of 7 min at 72° C. The PCR products were sizefractionated in 1% agarose gels and were visualized by ethidium bromidestaining. The results of these PCR amplifications are presented in Table2. The analysis of the amplification products revealed that BVH-MC2 (SEQID NO: 1) gene was present in the genome of all of the 4 M. catarrhalisstrains tested. No such product was detected when the control E. coliDNA was submitted to identical PCR amplifications with theseoligonucleotide primers.

Sequencing of additional BVH-MC2 genes from other strains confirmed thehigh level of molecular conservation of this gene among M. catarrhalisstrains. The respective coding region of M. catarrhalis BVH-MC2 genefrom strains ETSU 658 (SEQ ID NO: 3), ETSU T-25 (SEQ ID NO: 5), and M-12(SEQ ID NO: 7) were amplified by PCR using the oligonucleotide primersDMAR544 and DMAR545 as described above. PCR products were purified fromagarose gel using a QIAquick gel extraction kit from QIAgen followingthe manufacturer's instructions (Chatsworth, Calif.) and the DNA insertwere sequenced (Taq Dye Deoxy Terminator Cycle Sequencing kit, ABI,Foster City, Calif.). The total sequence could be obtained in the samemanner as in Example 1. The predicted amino acid sequences from strainsETSU C-2 (SEQ ID NO: 2), ETSU 658 (SEQ ID NO: 4), ETSU T-25 (SEQ ID NO:6), and M-12 (SEQ ID NO: 8) were respectively presented in the followingFIGS. 2, 4, 6, and 8. The FIGS. 15 and 16 respectively depict theconsensus nucleotide and predicted amino acid sequences established forM. catarrhalis BVH-MC2. Pairwise comparison of the BVH-MC2 predictedpolypeptide sequences revealed 100% identity. This latter result clearlydemonstrates the high level of molecular conservation of BVH-MC2polypeptides among M. catarrhalis isolates.

TABLE 2 Identification of M. catarrhalis genes by PCR amplification.Strain Identification by PCR amplification of Identification BVH-MC2BVH-MC3 BVH-MC4 BVH-MC5 ETSU C-2 + + + + ETSU 658 + + + + ETSUT-25 + + + + M-12 + + + + E. coil − − − −

EXAMPLE 2

This example illustrates the cloning and molecular characteristics ofBVH-MC3 gene and corresponding polypeptide.

The coding region of M. catarrhalis BVH-MC3 (SEQ ID NO: 9) gene wasamplified by PCR (DNA Thermal Cycler GeneAmp PCR system 2400 PerkinElmer, San Jose, Calif.) from genomic DNA of M. catarrhalis strain ETSUC-2 using the following oligos that contained base extensions for theaddition of restriction sites NdeI (CATATG) and XhoI (CTCGAG): DMARS92and DMAR593, which are presented in Table 1. The methods used forcloning BVH-MC3 into an expression vector and sequencing are similar tothe methods described in Example 1.

It was determined that the open reading frame (ORF) which codes forBVH-MC3 contains 1656-bp and encodes a 551 amino acid residuespolypeptide with a predicted pI of 4.68 and a predicted molecular massof 58910.13 Da. Analysis of the predicted amino acid residues sequence(SEQ ID NO: 10) using the Spscan software (Wisconsin Sequence AnalysisPackage; Genetics Computer Group) suggested the existence of a 46 aminoacid residues signal peptide (MSLINKLNERITPHVLTSIKINQDGDNADKSNLLTAFYTIFAGRLSN) (SEQ ID NO:32), which ends with acleavage site located between an asparagine and a glutamic acidresidues.

The BVH-MC3 gene was shown to be present after PCR amplification usingthe oligonucleotide primers DMAR592 and DMAR593 in the 4 M. catarrhalisstrains tested (Table 2). The methods used for PCR amplification of theBVH-MC3 gene were similar to the methods presented in Example 1. No suchproduct was detected when the control E. coli DNA was submitted toidentical PCR amplification with these oligonucleotide primers.

EXAMPLE 3

This example illustrates the cloning and molecular characteristics ofBVH-MC4 gene and corresponding polypeptide.

The coding region of M. catarrhalis BVH-MC4 (SEQ ID NO: 11) gene wasamplified by PCR (DNA Thermal Cycler GeneAmp PCR system 2400 PerkinElmer, San Jose, Calif.) from genomic DNA of M. catarrhalis strain ETSUC-2 using the following oligos that contained base extensions for theaddition of restriction sites NdeI (CATATG) and XhoI (CTCGAG): RIOS71and RIOS72, which are presented in Table 1. The methods used for cloningBVH-MC4 into an expression vector and sequencing are similar to themethods described in Example 1.

It was determined that the open reading frame (ORF) which codes forBVH-MC4 contains 1251-bp and encodes a 416 amino acid residuespolypeptide with a predicted pI of 4.84 and a predicted molecular massof 46125.11 Da. Analysis of the predicted amino acid residues sequence(SEQ ID NO: 12) using the Spscan software (Wisconsin Sequence AnalysisPackage; Genetics Computer Group) suggested the existence of a 42 aminoacid residues signal peptide(MDTKHIQQNWLLPDGVADVLFTDAQKQESLRDALLFVLTAHG) (SEQ ID SEQ:33), which endswith a cleavage site located between a glycine and a tyrosine residues.

The BVH-MC4 gene was shown to be present after PCR amplification usingthe oligonucleotide primers RIOS71 and RIOS72 in the 4 M. catarrhalisstrains tested (Table 2). The methods used for PCR amplification of theBVH-MC4 gene were similar to the methods presented in Example 1. No suchproduct was detected when the control E. coli DNA was submitted toidentical PCR amplification with these oligonucleotide primers.

EXAMPLE 4

This example illustrates the cloning and molecular characteristics ofBVH-MC5 gene and corresponding polypeptide.

The coding region of M. catarrhalis BVH-MCS (SEQ ID NO: 13) gene wasamplified by PCR (DNA Thermal Cycler GeneAmp PCR system 2400 PerkinElmer, San Jose, Calif.) from genomic DNA of M. catarrhalis strain ETSUC-2 using the following oligos that contained base extensions for theaddition of restriction sites NdeI (CATATG) and XhoI (CTCGAG): RIOS59and RIOS60, which are presented in Table 1. The methods used for cloningBVH-MC5 into an expression vector and sequencing are similar to themethods described in Example 1.

It was determined that the open reading frame (ORF) which codes forBVH-MC5 contains 639-bp and encodes a 212 amino acid residuespolypeptide with a predicted pI of 7.45 and a predicted molecular massof 24020.08 Da. Analysis of the predicted amino acid residues sequence(SEQ ID NO: 14) using the Spscan software (Wisconsin Sequence AnalysisPackage; Genetics Computer Group) suggested the existence of a 60 aminoacid residues signal peptide(MNNFVYQLQSFWYELNQVNRHTIAQSPKYIQLTVLGLIVMIIGIFGWLLAIL PTIQKLNA) (SEQ IDNO:34), which ends with a cleavage site located between two alanineresidues.

The BVH-MC5 gene was shown to be present after PCR amplification usingthe oligonucleotide primers RIOS59 and RIOS60 in the 4 M. catarrhalisstrains tested (Table 2). The methods used for PCR amplification of theBVH-MC5 gene were similar to the methods presented in Example 1. No suchproduct was detected when the control E. coli DNA was submitted toidentical PCR amplification with these oligonucleotide primers.

EXAMPLE 5

This example illustrates the cloning of M. catarrhalis genes in CMVplasmid pCMV-GH.

The DNA coding regions of a M. catarrhalis polypeptides were inserted inphase downstream of a human growth hormone (hGH) gene which was underthe transcriptional control of the cytomegalovirus (CMV) promotor in theplasmid vector pCMV-GH (Tang et al., Nature, 1992, 356:152). The CMVpromotor is non-functional plasmid in E. coli cells but active uponadministration of the plasmid in eukaryotic cells. The vector alsoincorporated the ampicillin resistance gene.

The coding regions of BVH-MC2 (SEQ ID NO: 1), BVH-MC3 (SEQ ID NO: 9),BVH-MC4 (SEQ ID NO: 11), and BVH-MC5 (SEQ ID NO: 13) genes without theirleader peptide regions were amplified by PCR (DNA Thermal CyclerGeneAmp® PCR system 2400 Perkin Elmer, San Jose, Calif.) from genomicDNA of M. catarrhalis strain ETSU C-2 using oligonucleotide primers thatcontained base extensions for the addition of restriction sites BamHI(GGATCC), BglII (AGATCT), SalI (GTCGAC), or HindIII (AAGCTT) which aredescribed in Table 1. The PCR products were purified from agarose gelusing a QIA® quick gel extraction kit from QIAgen® (Chatsworth, Calif.),digested with restriction enzymes (Amersham Pharmacia Biotech, Inc, Baied'Urfe, Canada). The pCMV-GH vector (Laboratory of Dr. Stephen A.Johnston, Department of Biochemistry, The University of Texas, Dallas,Tex.) was digested with BamHI, BglII, SalI, or HindIII and purified fromagarose gel using the QIA® quick gel extraction kit from QIAgen®(Chatsworth, Calif.). The digested DNA fragments were ligated to thedigested pCMV-GH vector to create the hGH-BVH-MC2, hGH-BVH-MC3,hGH-BVH-MC4, and hGH-BVH-MC5 fusion polypeptides under the control ofthe CMV promoter. The ligated products were transformed into E. colistrain DH5 α [φ80dlacZΔM15 Δ(lacZYA-argF)U169 endA1 recA1hsdR17(r_(K)-m_(K)+) deoR thi-1 supE44 λ⁻gyrA96 relA1](Gibco BRL,Gaithersburg, Md.) according to the method of Simanis (Hanahan, D. DNACloning, 1985, D.M. Glover (ed), pp. 109-135). The recombinant pCMVplasmids were purified using a QIAgen® kit (Chatsworth, Calif.) and thenucleotides sequence of the DNA inserts were verified by DNA sequencing.

EXAMPLE 6

This example illustrates the use of DNA to elicit an immune response toM. catarrhalis polypeptide antigens.

A group of 8 female BALB/c mice (Charles River, St-Constant, Quėbec,Canada) are immunized by intramuscular injection of 100 μl three timesat two- or three-week intervals with 50 μg of recombinant pCMV-GHencoding BVH-MC2 (SEQ ID NO: 1), BVH-MC3 (SEQ ID NO: 9), BVH-MC4 (SEQ IDNO: 11), and BVH-MC5 (SEQ ID NO: 13) genes in presence of 50 μg ofgranulocyte-macrophage colony-stimulating factor (GM-CSF)-expressingplasmid pCMV-GH-GM-CSF (Laboratory of Dr. Stephen A. Johnston,Department of Biochemistry, The University of Texas, Dallas, Tex.). Ascontrol, a group of mice are injected with 50 μg of pCMV-GH in thepresence of 50 μg of pCMV-GH-GM-CSF. Blood samples are collected fromthe orbital sinus prior to each immunization and seven days followingthe third injection and serum antibody responses are determined by ELISAusing the corresponding His-Tag labeled M. catarrhalis recombinantpolypeptides as coating antigen. The production and purification ofthese His-tagged labeled M. catarrhalis recombinant polypeptides arepresented in Example 7.

EXAMPLE 7

This example illustrates the production and purification of M.catarrhalis recombinant polypeptides.

The recombinant pET21b(+) plasmid with BVH-MC2 (SEQ ID NO: 1), BVH-MC3(SEQ ID NO: 9), BVH-MC4 (SEQ ID NO: 11), and BVH-MC5 (SEQ ID NO: 13)genes were used to transform by electroporation (Gene Pulser® IIapparatus, BIO-RAD Labs, Mississauga, Canada) E. coli strain AD494 (DE3)[Δara-leu7697 ΔlacX74 ΔphoA PvuII phoR ΔmalF3 F′[lac⁺(lacl^(q)) pro]trxB::Kan (DE3)] (Novagen®, Madison, Wis.). In this strain of E. coli,the T7 promotor controlling expression of the recombinant polypeptide isspecifically recognized by the T7 RNA polymerase (present on the λDE3prophage) whose gene is under the control of the lac promotor which isinducible by isopropyl-β-d-thio-galactopyranoside (IPTG). Thetransformant AD494(DE3)/rpET21b(+) was grown at 370° C. with agitationat 250 rpm in LB broth (peptone 10 g/L, yeast extract 5 g/L, NaCl 10g/L) containing 100 μg of carbenicillin (Sigma-Aldrich Canada Ltd.,Oakville, Canada) per ml until the A₆₀₀ reached a value of 0.5. In orderto induce the production of His-tagged M. catarrhalis recombinantpolypeptides, the cells were incubated for 3 additional hours in thepresence of IPTG at a final concentration of 1 mM. Induced cells from a500 ml culture were pelleted by centrifugation and frozen at −70° C.

The purification of the recombinant polypeptides from the solublecytoplasmic fraction of IPTG-induced AD494(DE3)/rpET21b(+) was done byaffinity chromatography based on the properties of the His.Tag sequence(6 consecutive histidine residues) to bind to divalent cations (Ni²⁺)immobilized on the His.Bind metal chelation resin. Briefly, the pelletedcells obtained from a 500 mL culture induced with IPTG was resuspendedin lysis buffer (20 mM Tris, 500 mM NaCl, 10 mM imidazole, pH 7.9)containing 1 mM PMSF, sonicated and centrifuged at 12,000 X g for 20 mmto remove debris. The supernatant was deposited on a Ni-NTA agarosecolumn (Qiagen®, Mississauga, Ontario, Canada). The His-tagged labeledM. catarrhalis recombinant polypeptides were eluted with 250 mMimidazole-500 mM NaCl-20 mM Tris pH 7.9. The removal of the salt andimidazole from the sample was done by dialysis against PBS at 420 C. Thequantities of recombinant polypeptides obtained from the solublefraction of E. coli were estimated by MicroBCA (Pierce, Rockford, Ill.).

EXAMPLE 8

This example illustrates the reactivity of the His-tagged M. catarrhalisrecombinant polypeptides with human palatine tonsils and sera collectedfrom mice after immunization with M. catarrhalis antigenic preparations.

As shown in Table 3, BVH-MC2, BVH-MC3, and BVH-MC4 His-taggedrecombinant polypeptides were recognized in immunoblots by theantibodies present in the human palatine tonsils. It indicates thathumans, which are normally in contact with M. catarrhalis do developantibodies that are specific to these polypeptides. These particularhuman antibodies might be implicated in the protection against M.catarrhalis infection. In addition, immunoblots also revealed that seracollected from mice immunized with M. catarrhalis antigenic preparationenriched membrane polypeptides which induced significant lung clearancein a mouse model also developed antibodies that recognized BVH-MC2His-tagged recombinant polypeptides. These results indicate that thispolypeptide was present in M. catarrhalis antigenic preparation thatprotected mice against infection and that it induced antibodies thatreacted with the corresponding BVH-MC2 His-tagged recombinantpolypeptide.

TABLE 3 Reactivity in immunoblots of human palatine tonsils and seracollected from mice after immunization with M. catarrhalis antigenicpreparations with M. catarrhalis His-tagged fusion recombinantpolypeptides. Purified Apparent Reactivity in immunoblots withrecombinant molecular weight Human palatine polypeptide I.D.¹ (kDa)²tonsils³ Mouse sera⁴ BVH-MC2 50 + − BVH-MC3 70 + + BVH-MC4 40 + −BVH-MC5 20 − − ¹His-tagged recombinant polypeptides produced andpurified as described in Example 7 were used to perform the immunoblots.²Molecular weight of the His-tagged recombinant polypeptide wasestimated after SDS-PAGE. ³Human palatine tonsils were undiluted toperform the immunoblots. ⁴Mouse sera collected after immunization withM. catarrhalis antigenic preparations enriched membrane polypeptideswere pooled and diluted 1/500 to perform the immunoblots. These micewere protected against a M. catarrhalis challenge.

EXAMPLE 9

This example illustrates the accessibility to antibodies of the BVH-MC2,BVH-MC3, BVH-MC4, and BVH-MC5 polypeptides at the surface of M.catarrhalis strain.

Bacteria were grown in Brain Heart Infusion (BHI) broth containing 0.25%dextrose at 37° C. in a 8% CO₂ atmosphere to give an OD_(490 nm) of0.650 (˜10⁸ CFU/ml). Dilutions of anti-BVH-MC2, anti-BVH-MC3,anti-BVH-MC4, anti-BVH-MC5, or control sera were then added and allowedto bind to the cells, which were incubated for 2 h at 4° C. withrotation. Samples were washed 4 times in blocking buffer[phosphate-buffered saline (PBS) containing 2% bovine serum albumin(BSA)], and then 1 ml of goat fluorescein (FITC)-conjugated anti-mouseIgG Fc (gamma) fragment specific diluted in blocking buffer was added.After an additional incubation of 60 min at room temperature withrotation in the dark, samples were washed 4 times in blocking buffer andfixed with 0.25% formaldehyde in PBS buffer for 18 h at 4° C. Cells werewashed 2 times in PBS buffer and resuspended in 0.5 ml of PBS buffer.Cells were kept in the dark at 4° C. until analyzed by flow cytometry(Epics® XL; Beckman Coulter, Inc.). Flow cytometric analysis revealedthat BVH-MC2-, BVH-MC3-, BVH-MC4-, and BVH-MC5-specific antibodiesefficiently recognized their corresponding surface exposed epitopes onthe homologous (ETSU C-2) M. catarrhalis strain tested (Table 4). It wasdetermined that more than 70% of the 10,000 Moraxella cells analyzedwere labeled with the antibodies present in the specific sera. Theseobservations clearly demonstrate that these polypeptides are accessibleat the surface where they can be easily recognized by antibodies.Anti-M. catarrhalis antibodies were shown to play an important role inthe protection against M. catarrhalis infection.

TABLE 4 Evaluation of the attachment of BVH-MC2-, BVH-MC3-, BVH-MC4-,and BVH-MC5-specific antibodies at the surface of intact cells of M.catarrhalis strain ETSU-C2. Serum Identification Fluorescence Index² %of labeled cells³ Pool of BVH-MC2-specific 3.6 72.8 sera¹ Pool ofBVH-MC3-specific 7.5 82.8 sera Pool of BVH-MC4-specific 10.9 92.4 seraPool of BVH-MC5-specific 6.7 77.4 sera Pool of negative control 1 7.4sera⁴ Positive control serum⁵ 43.8 98.7 ¹The mice were injectedsubcutaneously five times at two-week intervals with 20 μg of purifiedrecombinant polypeptides mixed with 10 μg of QuilA ™ (an adjuvantcontaining saponins from the bark of Quillaja saponaria) adjuvant(Cedarlane Laboratories, Hornby, Canada). The sera were diluted 1/50.²The fluorescence index was calculated as the median fluorescence valueobtained after labeling the cells with an immune serum divided by thefluorescence value obtained for a control mouse serum. A fluorescencevalue of 1 indicated that there was no binding of antibodies at thesurface of intact Moraxella cells. ³% of labeled cells out of the 10,000cells analyzed. ⁴Sera collected from unimmunized or sham-immunized micewere pooled, diluted 1/50, and used as negative controls for this assay.⁵Serum obtained from a mouse immunized with 20 μg of purified outermembrane polypeptides was diluted 1/1000 and was used as a positivecontrol for the assay.

EXAMPLE 10

This example illustrates the bactericidal activities of anti-BVH-MC2mouse sera.

Bacteria were plated on chocolate agar plate and incubated at 37° C. ina 8% CO₂ atmosphere for 18 h. Bacterial cells were then resuspended inbacteriolysis buffer [10% Hanks' Balanced Salt Solution (HBSS) and 1%hydrolyzed casein, pH 7.3] to an OD_(490nm) of 0.25 and diluted to 8×10⁴CFU/ml. The bactericidal assay was performed by mixing 25 μl of thebacterial suspension with 50 μl of diluted heat-inactivated test serumand 15 μl of HBSS and incubating for 15 min at 37° C., 8% CO₂ withagitation (200 rpm). The rabbit complement-containing serum was thenadded to a final concentration of 10%, and the mixture was incubated foran additional 60 min at 37° C., 8% CO₂ with agitation (200 rpm) At theend of the incubation period, the number of viable bacteria wasdetermined by plating 10 μl of the assay mixture on chocolate agarplate. The plates were incubated at 37° C. in an 8% CO₂ atmosphere for18-24 h. The control consisted of bacteria incubated withheat-inactivated sera collected from mice before immunization and rabbitcomplement.

The % of lysis was determined by the following mathematical formula:

$100 - \left\lbrack {\frac{A}{B} \times 100} \right\rbrack$

-   A=CFU obtained when the bacteria were incubated with immune sera-   B=CFU obtained with pre-bleed sera

The M. catarrhalis strain ETSU 658 was used to evaluate the bactericidalactivity of the sera. Percentage of lysis of 71.3 was determined formouse sera collected after immunization with purified recombinantBVH-MC2 polypeptide (SEQ ID NO: 2) (Table 5).

TABLE 5 Evaluation of the bactericidal activities of anti- BVH-MC2 mousesera. Identification Bactericidal titer % of lysis Pool of BVH-MC2- 1/3571.3 specific sera¹ Positive control 1/35 92.7 serum² ¹The mice wereinjected subcutaneously five times at two-week intervals with 20 μg ofpurified recombinant polypeptides mixed with 10 μg of QuilA ™ adjuvant(Cedarlane Laboratories, Hornby, Canada). ²Serum obtained from a mouseimmunized with 20 μg of purified outer membrane polypeptides was diluted1/35 and was used as a positive control for the assay.

EXAMPLE 11

This example illustrates the bactericidal activities of anti-BVH-MC3mouse sera.

Bacteria were plated on chocolate agar plates and incubated at 37° C. ina 8% CO₂ atmosphere for 18 h. Bacterial cells were then resuspended inbacteriolysis buffer [10% Hanks' Balanced Salt Solution (HBSS) and 1%hydrolyzed casein, pH 7.3] to an OD_(490nm) of 0.25 and diluted to 8×10⁴CFU/ml. The bactericidal assay was performed by mixing 25 μl of thebacterial suspension with 50 μl of diluted heat-inactivated test serumand 15 μl of HBSS and incubating for 15 min at 37° C., 8% CO₂ withagitation (200 rpm). The rabbit complement-containing serum was thenadded to a final concentration of 10%, and the mixture was incubated foran additional 60 min at 37° C., 8% CO₂ with agitation (200 rpm) At theend of the incubation period, the number of viable bacteria wasdetermined by plating 10 μl of the assay mixture on chocolate agarplate. The plates were incubated at 37° C. in an 8% CO₂ atmosphere for18-24 h. The control consisted of bacteria incubated withheat-inactivated sera collected from mice before immunization and rabbitcomplement. The M. catarrhalis strain ETSU 658 was used to evaluate thebactericidal activity of the sera. The % of lysis was determined by thefollowing mathematical formula:

$100 - \left\lbrack {\frac{A}{B} \times 100} \right\rbrack$

-   A=CFU obtained when the bacteria were incubated with immune sera-   B=CFU obtained with pre-bleed sera

Bactericidal antibodies were found to be present in the sera collectedfrom the 7 mice that were immunized with the purified recombinantBVH-MC3 polypeptide (Table 6). No bactericidal activity were recorded inthe sera collected from control mice (data not shown).

TABLE 6 Evaluation of the bactericidal activity of anti- BVE-MC3 mousesera. Serum identification¹ % of lysis S1² 33.3 S2 67.9 S3 89.6 S4 66.2S5 78.0 S6 90.1 S7 37.1 Positive control serum² 77.3 ¹The mice S1 to S7were injected subcutaneously five times at two-week intervals with 20 μgof purified recombinant polypeptide mixed with 10 μg of QuilA ™ adjuvant(Cedarlane Laboratories, Hornby, Canada) ²Each mouse serum collectedfrom BVR-MC3 immunized mouse were diluted 1/50. ³Serum obtained from amouse immunized with 20 μg of purified outer membrane polypeptides wasdiluted 1/50 and was used as a positive control for the assay.

EXAMPLE 12

This example illustrates the protection of mice against M. catarrhalisinfection induced by immunization with purified recombinant BVH-MC3polypeptide.

Groups of female BALB/c mice (Charles River) were immunizedsubcutaneously five times at two-week intervals with 20 μg of affinitypurified His-tagged M. catarrhalis recombinant BVH-MC3 polypeptide inpresence of 10% of QuilA adjuvant (Cedarlane Laboratories Ltd, Hornby,Canada) or, as control, with QuilA adjuvant alone in PBS. Blood sampleswere collected from the orbital sinus on day 0, 14, 28, 42, and 56 priorto each immunization and 14 days (day 70) following the fifth injection.One week later the mice were challenged intrapulmonary withapproximately 1×10⁶ CFU of the M. catarrhalis strain ETSU 658. Samplesof the M. catarrhalis challenge inoculum were plated on chocolate agarplates to determine the CFU and to verify the challenge dose. Mice werekilled by an intraperitoneal injection of sodium pentobarbital(Euthanyl™) 5 h after infection. The intact lungs were excised andhomogenised in a tissue homogeniser. The lung homogenate were assessedfor bacterial clearance by plating the serial dilutions for CFUdetermination. The % of clearance was determined by the followingmathematical formula:

$100 - \left\lbrack {\frac{A}{B} \times 100} \right\rbrack$

-   A=CFU obtained with the mice immunized with the BVH-MC3 polypeptide-   B=CFU obtained in the control mice

As shown in Table 7, a reduction of 54% in the number of living bacteriawere determined for mice immunized with BVH-MC3 polypeptidecomparatively to mice in the control group. Thus, immunization withrecombinant BVH-MC3 polypeptide promoted rapid clearance of anheterologous strain of M. catarrhalis from the lungs of the mice.

TABLE 7 Pulmonary clearance of Moraxella catarrhalis by mice immunizedwith purified recombinant BVH-MC3 polypeptide Bacterial recoveryBacterial recovery from control group from BVH-MC3 group Bacterial(CFU/ml of lung (CFU/ml of lung clearance homogenate)^(a)homogenate)^(b) (%)^(c) 2.4 × 10⁵ ± 1.9 × 10⁵ 1.1 × 10⁵ ± 7.9 × 10⁴ 54^(a)Means ± standard deviations for six mice. ^(b)Means ± standarddeviations for seven mice. ^(c)Mice were challenged intrapulmonary with1 × 10⁶ CFU of bacteria, and viable bacteria were recovered from lung 5h after challenge. The % of clearance was calculated with themathematical formula described above.

1. An isolated polypeptide comprising the amino acid sequence set forthas SEQ ID NO:10, wherein the polypeptide is capable of eliciting immuneresponse in a host Moraxella catarrhalis and wherein the isolatedpolypeptide is capable of eliciting antibodies that specifically bindsto a polypeptide consisting of the amino acid sequence set forth as SEQID NO:10.
 2. The isolated polypeptide of claim 1 from which the signalpeptide amino acid sequence set forth as SEQ.ID.NO:32 is deleted.
 3. Apharmaceutical composition that is a vaccine comprising the isolatedpolypeptide according to either claim 1 or claim 2 and apharmaceutically acceptable carrier, diluent or adjuvant.
 4. Apharmaceutical composition that is a vaccine comprising (a) apharmaceutically acceptable carrier, diluent or adjuvant and (b) anisolated polypeptide that consists of an amino acid sequence at least90% identical to the amino acid sequence set forth as SEQ ID NO: 10,wherein the isolated polypeptide is capable of eliciting an immuneresponse in a host against Moraxella catarrhalis, and wherein theisolated polypeptide is capable of eliciting antibodies thatspecifically bind to a polypeptide consisting of the sequence set forthas SEQ ID NO:10.
 5. A pharmaceutical composition of that is a vaccinecomprising a) a pharmaceutically acceptable carrier, diluent or adjuvantand (b) an isolated polypeptide that comprises an amino acid sequence atleast 95% identical to the amino acid sequence set forth as SEQ IDNO:10, wherein the isolated polypeptide is capable of eliciting animmune response in a host against Moraxella catarrhalis, and wherein theisolated polypeptide is capable of eliciting antibodies thatspecifically bind to a polypeptide consisting of the sequence set forthas SEQ ID NO:10.
 6. The pharmaceutical composition of claim 5 whereinthe isolated polypeptide comprises an amino acid sequence at least 95%identical to the amino acid sequence set forth as SEQ ID NO:10 and fromwhich the signal peptide amino acid sequence set forth as SEQ ID NO:32is deleted.
 7. A kit comprising a polypeptide according to either claim1 or claim 2 for detection or diagnosis of Moraxella catarrhalisinfection.